SUMMARY-PROJECT 2-CHANG: During an immune response to microbial infection, a naive CD8+ T lymphocyte can give rise to terminal effector cells that provide acute host defense and functionally distinct subsets of memory cells that provide durable immunity. Generation of one such subset, tissue-resident memory cells (Trm), is now recognized to be essential for protection at mucosal and body surfaces. Exhaustion, the failure of immune cells to achieve or maintain optimal function, is an alternative outcome that occurs in the setting of chronic infections and cancer. Prior studies have advanced our understanding of how CD8+ T cells differentiate into Trm or exhausted cells, but the field faces several important challenges that this Project seeks to overcome. We propose to take an unbiased, `phenotypic marker-agnostic' approach exploiting an innovative sequencing approach that enables simultaneous measurement of proteins and the transcriptome in the same single cells to overcome the inherent limitations of prior studies that have performed transcriptional profiling at the bulk population level. We will utilize an in vivo shRNA-based approach enabling us to reiteratively evaluate the function of 50 genes simultaneously, thereby overcoming the slow pace of discovery resulting from traditional genetic knockout models that evaluate one gene at a time. Specifically, we will use these approaches to achieve the following goals: (1) Investigate the molecular heterogeneity and functional importance of Trm cells within and among tissues; (2) Elucidate the ontogeny of Trm cells by reconstructing the transcriptomic roadmap from the naive state through progressive states of differentiation, within and across tissue sites; (3) Identify early, functionally important molecular determinants of exhausted circulating and tissue-resident CD8+ T cells. A major strength of our proposal is the integrated and synergistic design that exploits the strength of the individual Project and Core Principal Investigators, generating datasets that inform multiple Projects. We envision that insights resulting from our studies may have important implications for the rational design of vaccines and for therapeutic interventions that reverse the exhausted state.